Blind Curettage Technique for Treatment of Mucous Cysts Associated with Heberden Nodes: Description of Operative Technique.

The standard treatment for mucous cysts with Heberden nodes is excision of the dorsolateral osteophytes and capsule of the distal interphalangeal joint or thumb interphalangeal joint, including the stalk of the cyst. The skin incision varies for cases depending upon the geometry. We propose a surgical method utilizing blind lateral approaches for treating such mucous cysts.

Subtotal Second Toe Transfer for Finger Reconstruction after Malignant Fibroblastic Tumor Resection.

The transposition of an adjacent finger following the loss of a finger due to a malignant tumor resection improves hand function. However, patients may not accept the resulting appearance of a three-finger hand. A 28-year-old male with a malignant fibroblastic tumor at the base of the ring finger underwent resection of the tumor, excising the phalanx and a portion of the metacarpal. He refused a ray amputation and subsequent fifth-finger transposition. Therefore, we reconstructed the defect with a long-vascularized subtotal second toe from the metacarpal neck to the middle phalanx base of the fourth finger. There was no tumor recurrence, and the patient was highly satisfied with hand function and cosmetic appearance at 3 years of follow-up.

The ELEANOR noncoding RNA expression contributes to cancer dormancy and predicts late recurrence of estrogen receptor-positive breast cancer.

The recurrence risk of estrogen receptor (ER)-positive breast cancer remains high for a long period of time, unlike other types of cancer. Late recurrence reflects the ability of cancer cells to remain dormant through various events, including cancer stemness acquisition, but the detailed mechanism is unknown. ESR1 locus enhancing and activating noncoding RNAs (ELEANORS) are a cluster of nuclear noncoding RNAs originally identified in a recurrent breast cancer cell model. Although their functions as chromatin regulators in vitro are well characterized, their roles in vivo remain elusive. In this study, we evaluated the clinicopathologic features of ELEANORS, using primary and corresponding metastatic breast cancer tissues. The ELEANOR expression was restricted to ER-positive cases and well-correlated with the ER and progesterone receptor expression levels, especially at the metastatic sites. ELEANORS were detected in both primary and metastatic tumors (32% and 29%, respectively), and frequently in postmenopausal cases. Interestingly, after surgery, patients with ELEANOR-positive primary tumors showed increased relapse rates after, but not within, 5 years. Multivariate analysis showed that ELEANORS are an independent recurrence risk factor. Consistently, analyses with cell lines, mouse xenografts, and patient tissues revealed that ELEANORS upregulate a breast cancer stemness gene, CD44, and maintain the cancer stem cell population, which could facilitate tumor dormancy. Our findings highlight a new role of nuclear long noncoding RNAs and their clinical potential as predictive biomarkers and therapeutic targets for late recurrence of ER-positive breast cancer.

Environmentally contingent control of Candida albicans cell wall integrity by transcriptional regulator Cup9.

The fungal pathogen Candida albicans is surrounded by a cell wall that is the target of caspofungin and other echinocandin antifungals. Candida albicans can grow in several morphological forms, notably budding yeast and hyphae. Yeast and hyphal forms differ in cell wall composition, leading us to hypothesize that there may be distinct genes required for yeast and hyphal responses to caspofungin. Mutants in 27 genes reported previously to be caspofungin hypersensitive under yeast growth conditions were all caspofungin hypersensitive under hyphal growth conditions as well. However, a screen of mutants defective in transcription factor genes revealed that Cup9 is required for normal caspofungin tolerance under hyphal and not yeast growth conditions. In a hyphal-defective efg1Δ/Δ background, Cup9 is still required for normal caspofungin tolerance. This result argues that Cup9 function is related to growth conditions rather than cell morphology. RNA-seq conducted under hyphal growth conditions indicated that 361 genes were up-regulated and 145 genes were down-regulated in response to caspofungin treatment. Both classes of caspofungin-responsive genes were enriched for cell wall-related proteins, as expected for a response to disruption of cell wall integrity and biosynthesis. The cup9Δ/Δ mutant, treated with caspofungin, had reduced RNA levels of 40 caspofungin up-regulated genes, and had increased RNA levels of 8 caspofungin down-regulated genes, an indication that Cup9 has a narrow rather than global role in the cell wall integrity response. Five Cup9-activated surface-protein genes have roles in cell wall integrity, based on mutant analysis published previously (PGA31 and IFF11) or shown here (ORF19.3499, ORF19.851, or PGA28), and therefore may explain the hypersensitivity of the cup9Δ/Δmutant to caspofungin. Our findings define Cup9 as a new determinant of caspofungin susceptibility.

Sequence-dependent nucleosome formation in trinucleotide repeats evaluated by in vivo chemical mapping.

Trinucleotide repeat sequences (TRSs), consisting of 10 unique classes of repeats in DNA, are members of microsatellites and abundantly and non-randomly distributed in many eukaryotic genomes. The lengths of TRSs are mutable, and the expansions of several TRSs are implicated in hereditary neurological diseases. However, the underlying causes of the biased distribution and the dynamic properties of TRSs in the genome remain elusive. Here, we examined the effects of TRSs on nucleosome formation in vivo by histone H4-S47C site-directed chemical cleavages, using well-defined yeast minichromosomes in which each of the ten TRS classes resided in the central region of a positioned nucleosome. We showed that (AAT)12 and (ACT)12 act as strong nucleosome-promoting sequences, while (AGG)12 and (CCG)12 act as nucleosome-excluding sequences in vivo. The local histone binding affinity scores support the idea that nucleosome formation in TRSs, except for (AGG)12, is mainly determined by the affinity for the histone octamers. Overall, our study presents a framework for understanding the nucleosome-forming abilities of TRSs.

Use of Sponge-Foam Inserts in Compression Bandaging of Non-Healing Venous Leg Ulcers.

Objective: Venous leg ulcers (VLUs) caused by chronic venous insufficiency are difficult to treat. Outcomes after compression therapy and the current standard of care often used in conjunction with other options vary widely. We examined the effects of foam inserts on sub-bandage pressures in patients with VLUs and compared use of foam inserts in elastic and inelastic compression bandaging. Methods: Six patients (≥20 years old) with VLUs and skin perfusion pressure >40 mmHg were included. Each patient underwent weekly treatment regimens of debridement, dressing changes, and dual sponge-insert application followed by elastic (n=3) or inelastic (n=3) compression bandaging. The median resting sub-bandage pressures of the ulcer beds, wound sizes, and healing percentages were recorded. Wound beds were biopsied before and after treatment for histological assessment. Nine healthy volunteers served as controls during preliminary testing. Results: With proper sub-bandage pressures (>35 mmHg), the average healing time was 88.0±66 days, which was shorter than anticipated (i.e., ≥6 months). Combining large and local sponge-foam inserts increased sub-bandage pressures regardless of the compression bandage selected, with marked improvements seen in deeper wounds. Conclusion: Layering one or two sponge-foam inserts beneath compression bandages facilitates uniform and optimal wound-bed pressure, which accelerates the healing of VLUs.

Improved Methods for Preparing the Telomere Tethering Complex Bqt1-Bqt2 for Structural Studies.

In eukaryotes, chromosome ends (telomeres) are tethered to the inner nuclear membrane. During the early stages of meiosis, telomeres move along the nuclear membrane and gather near the spindle-pole body, resulting in a bouquet-like arrangement of chromosomes. This chromosomal configuration appears to be widely conserved among eukaryotes, and is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In fission yeast, the Bqt1-Bqt2 protein complex plays a key role in tethering the telomere to the inner nuclear membrane. However, the structural details of the complex required to clarify how telomeres are gathered near the spindle-pole body remain enigmatic. Previously, we devised a preparation procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, in which a SUMO tag was fused to the N-terminus of the Bqt1 protein. This allowed us to purify the Bqt1-Bqt2 complex from the soluble fraction. In the present study, we found that a maltose-binding protein homolog, Athe_0614, served as a better fusion partner than the SUMO protein, resulting in the marked increase in the solubility of the Bqt1-Bqt2 complex. The Athe_0614 fusion partner may open up new avenues for X-ray crystallographic analyses of the structure of the Bqt1-Bqt2 complex.

Nucleosome destabilization by nuclear non-coding RNAs.

In the nucleus, genomic DNA is wrapped around histone octamers to form nucleosomes. In principle, nucleosomes are substantial barriers to transcriptional activities. Nuclear non-coding RNAs (ncRNAs) are proposed to function in chromatin conformation modulation and transcriptional regulation. However, it remains unclear how ncRNAs affect the nucleosome structure. Eleanors are clusters of ncRNAs that accumulate around the estrogen receptor-α (ESR1) gene locus in long-term estrogen deprivation (LTED) breast cancer cells, and markedly enhance the transcription of the ESR1 gene. Here we detected nucleosome depletion around the transcription site of Eleanor2, the most highly expressed Eleanor in the LTED cells. We found that the purified Eleanor2 RNA fragment drastically destabilized the nucleosome in vitro. This activity was also exerted by other ncRNAs, but not by poly(U) RNA or DNA. The RNA-mediated nucleosome destabilization may be a common feature among natural nuclear RNAs, and may function in transcription regulation in chromatin.

Exposed Artificial Plate Covered With Perifascial Areolar Tissue as a Nonvascularized Graft.

Perifascial areolar tissue (PAT) is a loose connective tissue on deep fascia, such as on the groin, thigh, or temporal region, which has abundant vascular plexus and mesenchymal stem cells. Nonvascularized PAT grafts can survive even on hypovascular wound beds. Therefore, PAT grafting is a possible alternative to conventional flap surgery to cover exposed bone or artifacts. In this article, we describe 2 cases of PAT grafting for the treatment of skin ulcers with exposed bone and artificial plate after mandible reconstruction. After negative-pressure wound therapy, PAT was used to covering exposed artificial plate for both cases, and a skin graft onto the PAT graft was performed in 1 case. The ulcers improved in both cases without recurrence. The gold-standard treatment of intractable ulcers and fistulas with an exposed tendon, bone, or artifact is coverage by a well-vascularized skin flap. However, PAT grafting has advantages in similar situations, occasionally together with skin grafting and/or negative-pressure wound therapy, because it is technically simple and less invasive.

Split-skin Paddle Anterolateral Thigh Flap for Reconstruction of Giant Dermatofibrosarcoma Protuberans in Groin.

Dermatofibrosarcoma protuberans (DFSP) is a slow-growing superficial sarcoma. Due to its high late local recurrence, thin skin graft is usually recommended for reconstruction after resection of the tumor. In this report, we present a case of giant DFSP in the groin region of young woman. Reconstruction of large groin defect after DFSP resection was performed by "split-skin paddle anterolateral thigh flap" instead of skin graft considering that the patient was a 29-year-old woman. This method enabled the primary closure of the donor site and provided the positive functional and esthetic outcomes. In the present case, the surgical scar is less conspicuous and the patient can climb and descend stairs without any trouble at 4 years after the surgery. With careful monitoring of the tumor recurrence, this technique may become a reliable reconstruction option for patients with large groin defect after resection of the malignant tumor.

Novel genetic tools for probing individual H3 molecules in each nucleosome.

In eukaryotes, genomic DNA is packaged into the nucleus together with histone proteins, forming chromatin. The fundamental repeating unit of chromatin is the nucleosome, a naturally symmetric structure that wraps DNA and is the substrate for numerous regulatory post-translational modifications. However, the biological significance of nucleosomal symmetry until recently had been unexplored. To investigate this issue, we developed an obligate pair of histone H3 heterodimers, a novel genetic tool that allowed us to modulate modification sites on individual H3 molecules within nucleosomes in vivo. We used these constructs for molecular genetic studies, for example demonstrating that H3K36 methylation on a single H3 molecule per nucleosome in vivo is sufficient to restrain cryptic transcription. We also used asymmetric nucleosomes for mass spectrometric analysis of dependency relationships among histone modifications. Furthermore, we extended this system to the centromeric H3 isoform (Cse4/CENP-A), gaining insights into centromeric nucleosomal symmetry and structure. In this review, we summarize our findings and discuss the utility of this novel approach.

Correction to: Novel genetic tools for probing individual H3 molecules in each nucleosome.

In the original publication, Fig. 1 was incorrectly published. The amino acid sequence was shifted to the left relative to the rest of the diagram in the published version and the corrected figure is given here.

An asymmetric centromeric nucleosome.

Nucleosomes contain two copies of each core histone, held together by a naturally symmetric, homodimeric histone H3-H3 interface. This symmetry has complicated efforts to determine the regulatory potential of this architecture. Through molecular design and in vivo selection, we recently generated obligately heterodimeric H3s, providing a powerful tool for discovery of the degree to which nucleosome symmetry regulates chromosomal functions in living cells (Ichikawa et al., 2017). We now have extended this tool to the centromeric H3 isoform (Cse4/CENP-A) in budding yeast. These studies indicate that a single Cse4 N- or C-terminal extension per pair of Cse4 molecules is sufficient for kinetochore function, and validate previous experiments indicating that an octameric centromeric nucleosome is required for viability in this organism. These data also support the generality of the H3 asymmetric interface for probing general questions in chromatin biology.

Parallel mapping with site-directed hydroxyl radicals and micrococcal nuclease reveals structural features of positioned nucleosomes in vivo.

Micrococcal nuclease (MNase) has been widely used for analyses of nucleosome locations in many organisms. However, due to its sequence preference, the interpretations of the positions and occupancies of nucleosomes using MNase have remained controversial. Next-generation sequencing (NGS) has also been utilized for analyses of MNase-digests, but some technical biases are commonly present in the NGS experiments. Here, we established a gel-based method to map nucleosome positions in Saccharomyces cerevisiae, using isolated nuclei as the substrate for the histone H4 S47C-site-directed chemical cleavage in parallel with MNase digestion. The parallel mapping allowed us to compare the chemically and enzymatically cleaved sites by indirect end-labeling and primer extension mapping, and thus we could determine the nucleosome positions and the sizes of the nucleosome-free regions (or nucleosome-depleted regions) more accurately, as compared to nucleosome mapping by MNase alone. The analysis also revealed that the structural features of the nucleosomes flanked by the nucleosome-free region were different from those within regularly arrayed nucleosomes, showing that the structures and dynamics of individual nucleosomes strongly depend on their locations. Moreover, we demonstrated that the parallel mapping results were generally consistent with the previous genome-wide chemical mapping and MNase-Seq results. Thus, the gel-based parallel mapping will be useful for the analysis of a specific locus under various conditions.

A synthetic biology approach to probing nucleosome symmetry.

The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.

Takotsubo Cardiomyopathy after Head and Neck Reconstructive Surgery.

Takotsubo cardiomyopathy (TCM) is a form of transient heart failure that clinically mimics acute coronary syndrome and is characterized by left ventricular wall motion abnormalities. The pathophysiology of TCM is not well established. TCM is often preceded by emotional or physical stress and may occur after surgery. We present 3 cases of TCM occurring after head and neck reconstructive surgery. Echocardiography plays a central role in the diagnosis of TCM. Left ventricular wall motion abnormalities extend beyond the territory of a single coronary artery. Coronary angiography and cardiac computed tomography can demonstrate the absence of coronary atherosclerosis and are useful for confirming the diagnosis of TCM. Particularly after reconstructive surgery, it is necessary to carefully monitor fluid replacement to avoid dehydration, which may compromise flap blood flow, although congestive heart failure is the most common complication of TCM. It is important to encourage ambulation as soon as possible, while considering the degree of cardiac impairment.